Bordetella strains expressing serotype 3 fimbriae

ABSTRACT

A Fim3-producing BPZE1 derivative with sufficiently stable fim3 expression to provide improved protection in mice against Fim3-only producing clinical  B. pertussis  isolates was developed. The fim3 expression in BPZElf3 did not alter the protective efficacy against Fim2+ strains, nor against strains that produce neither Fim2 nor Fim3.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a by-pass continuation under 35 U.S.C. 111(a)of international patent application number PCT/EP2018/078522 filed onOct. 18, 2018 which claims the priority of U.S. provisional patentapplication Ser. No. 62/574,068 filed on Oct. 18, 2017.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 10, 2018, isnamed 7056-0091_SL and is 1,833 bytes in size.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

Not applicable.

FIELD OF THE INVENTION

The invention relates generally to the fields of microbiology,immunology, vaccinology, sero-epidemiology, biochemistry and medicine.More particularly, the invention relates to live attenuated Bordetellastrains modified to express serotype 3 fimbriae and their use invaccines.

BACKGROUND

Whooping cough or pertussis is a severe respiratory disease that can belife-threatening, especially in young infants, and its incidence is onthe rise in several countries, despite a global vaccination coverageof >85%, according to the World Health Organization. However, it alsoaffects adolescents and adults, where symptoms are usually atypical, andtherefore the disease often remains undiagnosed in these age groups.Nevertheless, adolescents and adults, even if they remain asymptomatic,can transmit the causative agent Bordetella pertussis to young infantsbefore they are protected by the primary vaccination series. In fact, arecent wavelet analysis of B. pertussis infection in the US and the UK,combined with a phylodynamic analysis of clinical isolates showed thatasymptomatic transmission is the principle cause of the recent pertussisresurgence. In addition, asymptomatic B. pertussis infection may not beanodyne, as epidemiological evidence suggests that B. pertussisinfection may be related to auto-immune diseases, such as Celiacdisease, multiple sclerosis, and even Alzheimer's disease

Currently available whole-cell or acellular vaccines have been veryeffective in reducing the incidence of whooping cough after threeprimary vaccination doses. However, in contrast to prior infection withB. pertussis, they are much less effective in reducing asymptomaticcolonization, as shown in the recently established baboon model.Although vaccinated baboons were protected against pertussis diseaseupon experimental infection with B. pertussis, they could readily beinfected and transmit the organism to littermates, even in the absenceof symptoms, in contrast to convalescent baboons. Altogether theseobservations illustrate the shortcomings of currently availablevaccines, and call for new vaccines that protect both against diseaseand infection.

Based on the observation that the best way to protect against B.pertussis colonization is prior infection, a live attenuated vaccine hasbeen developed that can be administered by the nasal route, in order tomimic as much as possible natural infection without causing disease. Thevaccine strain, called BPZE1, lacks the gene coding for dermonecrotictoxin, produces genetically detoxified pertussis toxin and is deficientfor tracheal cytotoxin production by the replacement of the B. pertussisampG gene with the Escherichia coli ampG gene. BPZE1 has been shown tobe safe in pre-clinical models, including in severely immunocompromisedmice, and to be genetically stable after serial passages in vitro and invivo for at least 12 months. It protects mice against B. pertussischallenge after a single nasal administration, both via protective CD4+T cells and antibodies, and protection was shown to be long lived aftera single nasal vaccination. It also has recently been shown to reducenasopharyngeal infection by B. pertussis in baboons by 99.992% comparedto non-vaccinated baboons. BPZE1 has now successfully completed afirst-in-man phase I clinical trial and was found to be safe in humanadults, able to transiently colonize the human nasopharynx and to induceimmune responses to all tested antigens in all colonized individuals.

B. pertussis produces two serologically distinct fimbriae, composed ofeither Fim2 or Fim3 as the major fimbrial subunit. These fimbriae areinvolved in the attachment of the bacteria to respiratory epithelialcells. While BPZE1 produces only Fim2, it also produces hundreds ofother antigens (e.g., pertussis toxin, FHA, and pertactin). It thus hasbeen shown to induce significant protection against a wide array of B.pertussis clinical isolates, including those, which only produce Fim3.

SUMMARY

Described herein is the development of BPZE1f3, deposited with theCollection Nationale de Cultures de Microorganismes (CNCM, InstitutPasteur, 25 rue du Docteur Roux, F-75724 Paris Cedex 15, FRANCE) on Oct.11, 2017 under registration number CNCM I-5247, a B. pertussis strainderived from BPZE1 that produces both serotype 2 fimbriae (Fim2) andserotype 3 fimbriae (Fim3). Given that BPZE1 produces several hundreddifferent non-fimbriae antigens that could be targeted by immuneresponses, adding a single new antigen was not expected to have mucheffect on the protective effect of the bacteria. It was thussurprisingly discovered that vaccination with BPZElf3 significantlyimproved the protective effect against certain clinical isolates whichproduce Fim3 and not Fim2.

In the study described in the Examples section below, an intranasalmouse challenge model was used to examine the protective potential ofFim2-producing BPZE1 and Fim2-, 3-producing BPZElf3 to protect againstclinical isolates of different serotypes. Both vaccine strains appearedto induce significant protection against all examined clinical isolates.However, BPZElf3 provided significantly better protection than BPZE1against a clinical isolate that only produced Fim3, confirmingsero-specific protection to a certain degree.

A number of Fim2 and Fim3 subtypes have been identified. These includetwo Fim2 subtypes, Fim2-1, Fim2-2, which vary from each other by asingle amino acid difference. Fim2-1 carries an arginine at position174, whereas this is changed to a lysine in Fim2-2. The Fim3 subtypesare encoded by 6 different alleles. Fim3-2 differs from Fim3-1 by asingle amino acid substitution at position 87: Alanine and Glutamate forFim3-1 and Fim3-2, respectively. Fim3-3 carries, in addition to theGlutamate substitution at position 87, a change from Threonine in Fim3-1to Alanine in Fim3-3. The fim3-4 allele differs from fim3-1 only by asingle silent nucleotide polymorphism, whereas the other five allelesvary by three codons, each leading to one amino acid change in the majorfimbrial subunit. Given the minor sequence differences between thevarious subtypes, it is likely that BPZElf3 is protective against all ofthem.

To induce immune responses to fimbrial antigens, production of theseantigens must be sufficiently stable in live B. pertussis vaccinestrains. The stability of the Fim2 and Fim3 production deservesparticular attention, since phase variation from one serotype to anotherhas been described, especially during infection, and can be driven byvaccine pressure. This phase transition from high to low fimbrialproduction depends on the number of cytosines present in a C-stringwithin the fin promoter region. The number of cytosines within thisC-string may affect the distance between the −10 box of the fimpromoters and the binding site of BvgA, the transcriptional activatorrequired for the expression of fim and other B. pertussis virulencegenes. It has long been known that DNA regions with repeated base pairssequences, predominantly in C-strings, are particularly prone toadditions or deletions of a single base. Since BPZE1f3 was constructedby the addition of a single C:G base pair in a stretch of 13 C in thepromoter region to allow for fim3 expression, it was thought that thefim3 expression would be unstable. Unexpectedly, however, after severalpassages of BPZE1f3 through mice, 100% of the bacteria recovered afterthe first passage remained Fim3+, as well as Fim2+. During subsequentpassages (up to 3), close to 90% of the bacteria still expressed bothfim3 and fim2, indicating that fim expression was sufficiently stable toinduce serotype-specific immunity, as confirmed by the protective effectof BPZE1f3 against Fim3-only producing clinical isolates.

Accordingly, described herein is a live attenuated Bordetella strainengineered to stably produce Fim3, wherein the live attenuatedBordetella strain retains the ability to colonize a mammalian subject'slungs and induce a protective immune response against Bordetellainfection (e.g., the Bordetella strain designated BPZE1f3). The liveattenuated Bordetella strain can be one that also stably produces Fim2.The live attenuated Bordetella strains described herein can also berendered deficient in at least one (1, 2, or 3) of \ the followingvirulence factors: a functional pertussis toxin (PTX), a functionaldermonecrotic toxin (DNT), and a functional tracheal cytotoxin (TCT).

Also described herein are vaccines that include a live attenuatedBordetella strain engineered to stably produce Fim3 mentioned herein anda pharmaceutically acceptable carrier. The vaccine can be provided in asingle dosage form which includes at least 1×10⁶ (e.g., at least 1×10⁶,5×10⁶, or 1×10⁷) colony forming units (CFU) of the strain.

Further described herein are methods of protecting a mammalian subject(e.g., a human being) from developing pertussis, which include the stepof administering to the mammalian subject a vaccine including apharmaceutically acceptable carrier and a live attenuated Bordetellastrain engineered to stably produce Fim3, wherein the live attenuatedBordetella strain retains the ability to colonize a mammalian subject'slungs and induce a protective immune response against Bordetellainfection.

As used herein, a bacterial strain that “stably produces” an antigen isone that can be passaged at least once (e.g., 1, 2, 3, 4, 5 or moretimes) through a host animal without losing more than 50% (or more than60, 70, 80, 90, 95, 97, 98, or 99%) of the expression of that antigen.For example, an isolated Bordetella bacterial strain engineered tostably produce Fim3 is one that has been genetically modified to expressFim3, and retain at least 50% (e.g., 50, 60, 70, 80, 90, 95, 97, 98, or99%) of the expression of Fim-3 after being passaged through a mouse,e.g., by the methods described in the Examples section below.

Reference to a “functional” virulence factor means that a bacterialstrain possesses at least 50% of the enzymatic activity of thatvirulence factor compared to a the wild-type version of that virulencefactor. A bacterial strain that “has been rendered deficient in at leastone virulence factor” is a strain engineered to express less than 70,80, 90, 95, 96, 97, 98, or 99% of the enzymatic or functional activityof that virulence factor as compared to the parent strain from which iswas derived.

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs. Although methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention, suitable methods and materials aredescribed below. All publications, patents, and patent applicationsmentioned herein are incorporated by reference in their entirety. In thecase of conflict, the present specification, including definitions willcontrol. In addition, the particular embodiments discussed below areillustrative only and not intended to be limiting.

DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph showing the production of Fim2 by BPZE1 (in diamonds)and BPZElf3 (in squares).

FIG. 1B is a graph showing the production of Fim3 by BPZE1 (in diamonds)and BPZElf3 (in squares).

FIG. 2A is a graph showing the in vitro growth of BPZE1 (in diamonds)and BPZElf3 (in squares) in modified Stainer-Scholte medium.

FIG. 2B is a graph showing the in vitro growth of BPZE1 (in diamonds)and BPZElf3 (in squares) in fully synthetic Thijs medium.

FIG. 3 is a graph showing lung colonization of mice nasally inoculatedwith 10⁶ CFU of BPZE1 (in black) or BPZE1f3 (in grey), where thebacterial loads in the lungs were measured at the indicated time points.

FIGS. 4A-4E represent a series of graphs showing BPZE1- andBPZE1f3-induced protection against clinical B. pertussis isolates wheremice received nasally either 10⁵ CFU of BPZE1 (black bars) or BPZE1f3(grey bars), or were left untreated (white bars). Four weeks aftervaccination the mice were challenged with 10⁶ CFU of 1617pF1 (A), 403pF1(B), P134 (C), 1412pF1 (D) or 403pF3 (E). Three h (left part of thepanels, DO) or 7 days (right part of the panels, D7) after challenge,the bacterial loads in the lungs were measured and are presented asmeans and standard deviations of CFU. Three (for DO) or five (for D7)mice per group were used. ***, p<0.001.

FIG. 5 is a graph comparing BPZE1- and BPZE1f3-induced protectionagainst B. parapertussis where mice received nasally either 10⁶ CFU ofBPZE1 (black bars) or BPZE1f3 (grey bars), or were left untreated (whitebars). Two months after vaccination the mice were challenged with 10⁶CFU of B. parapertussis. Three h (left part of the panel, DO) or 7 days(right part of the panel, D7) after challenge, the bacterial loads inthe lungs were measured and are presented as means and standarddeviations of CFU. Three (for DO) or five (for D7) mice per group wereused. ***, p<0.001.

FIG. 6 is a graph showing the stability of Fim2 and Fim3 production byBPZE1f3, where BPZE1f3 was passaged three times in mice, and at eachpassage (P1 to P3), 94 colonies were analysed by whole-cell ELISA forthe presence of Fim2 (white bars) and Fim3 (black bars) using anti-Fim2and anti-Fim3 monoclonal antibodies.

DETAILED DESCRIPTION

Described herein is a Fim3-producing BPZE1 derivative with sufficientlystable fim3 expression to provide improved protection in mice againstFim3-only producing clinical B. pertussis isolates. The fim3 expressionin BPZE1f3 did not alter the protective efficacy against Fim2+ strains,nor against strains that produce neither Fim2 nor Fim3. The belowdescribed embodiments illustrate representative examples of thesemethods. Nonetheless, from the description of these embodiments, otheraspects of the invention can be made and/or practiced based on thedescription provided below.

General Methodology

Methods involving conventional microbiological, immunological, molecularbiological, and medical techniques are described herein. Microbiologicalmethods are described in Methods for General and Molecular Microbiology(3d Ed), Reddy et al., ed., ASM Press. Immunological methods aregenerally known in the art and described in methodology treatises suchas Current Protocols in Immunology, Coligan et al., ed., John Wiley &Sons, New York. Techniques of molecular biology are described in detailin treatises such as Molecular Cloning: A Laboratory Manual, 2nd ed.,vol. 1-3, Sambrook et al., ed., Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 2001; and Current Protocols in MolecularBiology, Ausubel et al., ed., Greene Publishing and Wiley-Interscience,New York. General methods of medical treatment are described in McPheeand Papadakis, Current Medical Diagnosis and Treatment 2010, 49thEdition, McGraw-Hill Medical, 2010; and Fauci et al., Harrison'sPrinciples of Internal Medicine, 17th Edition, McGraw-Hill Professional,2008.

Fim3-Producing Bordetella Strains

Bordetella species (e.g., Bordetella pertussis, Bordetellaparapertussis, and Bordetella bronchiseptica) that lack Fim3 expressioncan be engineered to produce Fim3 (e.g., Fim3-1, Fim3-2, Fim3-3, orFim3-4), and otherwise attenuated as described below. TheseFim3-producing bacteria might be used to treat and/or preventsymptomatic or asymptomatic respiratory tract infections caused byBordetella species as well as other conditions where BPZE1 was shown tobe effective (e.g., allergy and asthma). Bordetella strains engineeredto produce Fim3 might also be used to prevent transmission of Bordetellainfections. Attenuated, Fim2-/Fim3-producing Bordetella pertussis ispreferred for use in human subjects. Bordetella strains for use inmaking Fim3-producing bacteria can be isolated from natural sources(e.g., colonized subjects) or obtained from various culture collections.Bordetella strains that have been engineered to produce Fim3 can be madeby the methods described below.

Because insufficient attenuation of a pathogenic strain of Bordetellamight cause a pathological infection in a subject, it is preferred thatthe Bordetella strain engineered to produce Fim3 have lower levels ofother virulence factors. On the other hand, to ensure that theFim3-producing Bordetella strains are able to colonize a subject andexert a protective effect on respiratory tract inflammation, it must notbe overly attenuated. Attenuation might be achieved by mutating thestrain to reduce its production of one or more (e.g., 1, 2, 3, 4, 5 ormore) of the following: pertussis toxin (PTX), dermonecrotic toxin(DNT), tracheal cytotoxin (TCT), adenylate cyclase (AC),lipopolysaccharide (LPS), filamentous hemagglutinin (FHA), pertactin, orany of the bvg-regulated components. Methods for making such mutants aredescribed herein and in U.S. Pat. No. 9,119,804 and U.S. patentapplication Ser. No. 15/472,436. In the experiments presented below, aBordetella strain was engineered to produce Fim 3 that was deficient inDNT and TCT and produced genetically inactive PTX. It was able tocolonize the respiratory tract of and induce a protective immuneresponse in, subjects.

Formulations/Dosage/Administration

The Bordetella strains engineered to produce Fim 3 can be formulated asa vaccine for administration to a subject. A suitable number of livebacteria are mixed with a pharmaceutically suitable excipient or carriersuch as phosphate buffered saline solutions, distilled water, emulsionssuch as an oil/water emulsions, various types of wetting agents sterilesolutions and the like. In some cases the vaccine can be lyophilized andthen reconstituted prior to administration. The use of pharmaceuticallysuitable excipients or carriers which are compatible with mucosal(particularly nasal, bronchial, or lung) administration are preferredfor the purpose of colonizing the respiratory tract. See Remington'sPharmaceutical Sciences, a standard text in this field, and in USP/NF.

When formulated for mucosal administration, each dose of the vaccine caninclude a sufficient number of live Bordetella bacteria to result incolonization of the respiratory tract, e.g., approximately (i.e.,+/−50%) 5×10³ to 5×10⁹ bacteria, depending on the weight and age of themammal receiving it. For administration to human subjects, the dose caninclude approximately 1×10⁶, 5×10⁶, 1×10, 5×10⁷, 1×10⁸, 5×10⁸, 1×10⁹,5×10⁹, or 1×10¹⁰ live Fim3-producing Bordetella bacteria. The dose maybe given once or on multiple (2, 3, 4, 5, 6, 7, 8 or more) occasions atintervals of 1, 2, 3, 4, 5, or 6 days or 1, 2, 3, 4, 5, or 6 weeks, or1, 2, 3, 4, 5, 6, or 12 months. Generally, sufficient amounts of thevaccine are administered to result in colonization and the protectiveresponse. Additional amounts are administered after the inducedprotective response wanes.

Methods of Eliciting Immune Responses to Protect Against Pertussis

The vaccines described herein can be administered to a mammalian subject(e.g., a human being, a human child or neonate, a human adult, a humanbeing at high risk from developing complications from pertussis, a humanbeing with lung disease, and a human being that is or will becomeimmunosuppressed) by any suitable method that deposits the bacteriawithin the vaccine in the respiratory tract. For example, the vaccinesmay be administered by inhalation or intranasal introduction, e.g.,using an inhaler, a syringe, an insufflator, a spraying device, etc.While administration of a single dose of between 1×10⁴ to 1×10⁷ (e.g.,1×10⁴, 5×10⁴, 1×10⁵, 5×10⁵, 1×10⁶, 5×10⁶, or 1×10⁷+/−10, 20, 30, 40, 50,60, 70, 80, or 90%) live bacteria is typically sufficient to induceprotective immunity against developing a Bordetella infection such aspertussis, one or more (1, 2, 3, 4, or more) additional doses might beadministered in intervals of 4 or more days (e.g., 4, 5, 6, or 7 days;or 1, 2 3, 4, 5, 6, 7, or 8 weeks) until a sufficiently protectiveimmune response has developed. The development of a protective immuneresponse can be evaluated by methods known in the art such asquantifying Bordetella-specific antibody titers and measuring ofBordetella antigen-specific T cells responses (e.g., using an ELISPOTassay). In cases were a vaccine-induced protective immune response haswaned (e.g., after 1, 2, 3, 4, 5, 10 or more years from the lastvaccination) a subject may again be administered the vaccine in order toboost the anti-Bordetella immune response.

EXAMPLES

Materials and Methods

Culture Conditions

All B. pertussis strains were grown on Bordet Gengou (BG) agar with 10%(v/v) sheep blood, in modified Stainer Scholte (SS) medium underagitation as described (Imaizumi et al., Infect Immun 1983; 41:1138-43)or in fully synthetic Thijs medium (Thalen et al., J Biotechnol 1999;75:147-59). The media were supplemented with the appropriate antibiotics(100 ug/ml of streptomycin or 10 ug/ml of gentamycin for the strainscarrying pFUS2 BctA1).

Bacterial Strains

B. pertussis BPSM and BPZE1, as well as Bordetella parapertussis used inthis study have been described previously (Mielcarek et al., PLoS Pathog2006; 2:e65; Menozzi et al., Infect Immun 1994; 62:769-78). B. pertussisstrains B0403, B1412, B1617 and B0005 (strain 134 Pillmer) came from theRIVM collection (Bilthoven, The Netherlands). For counter-selectionpurposes some of the clinical isolates strains were electroporated withthe pFUS2 BctA1 suicide plasmid to acquire the gentamycin resistance asdescribed in Antoine et al. (J Mol Biol 2005; 351:799-809).Gentamycin-resistant derivatives after electroporation were checked byPCR to verify the site of insertion of the pFUS2 BctA1 vector into thechromosomal DNA and by ELISA to check the level of surface exposed Fim2and/or Fim3, as described below. Strain P134S was obtained by selectinga streptomycin derivative of B. pertussis B0005. Strain P134S carries,in addition to streptomycin resistance mediated by a mutation in therpsl gene, a mutation in thefimC gene leading to the loss of thefimbriae production. Escherichia coli SM10 (Simon et al., Bio/Technology1983; 1:784-91) was used for conjugation of the various plasmidconstructs into B. pertussis.

Construction of the Fim3-Positive BPZE1-Derivative BPZE1f3

To construct BPZE1f3, the 13 C stretch located in the promoter region ofthe fim3 gene of BPZE1, 75 bp upstream of the fim3 ATG codon, wasreplaced by a 14 C stretch in order to trigger the transcription offim3. The whole fim3 locus, containing the promoter region, was firstdeleted in the parental strain and then replaced by a fim3 locus with a14 C stretch. A 2265-bp PCR fragment encompassing the locus wasamplified by using the following oligonucleotides (SPfim3UP2:GAGCTCTTTACCGCGGCCGCCAGTTGTTCATCAATG and ASPfim3LO2:GGATCCATCATCGAGACCGACTGG) and cloned into the SacI and BamHI restrictionsites of a pBluescript II SK+ plasmid (Addgene). From resulting plasmid,a 904-bp fragment containing the whole locus was removed by SphIrestriction to obtain pSKfim3UPLO. The 1351-bp SacI-BamHI fragment ofpSKfim3UPLO was inserted into the SacI and BamHI sites of pJQ200mp18rpsL(Antoine, J. Mol. Biol. (2005) 351, 799-809). The recombinant plasmidwas then used for double homologous recombination in BPZE1 usingconjugation as described previously (Mielcarek et al., PLoS Pathog 2006;2:e65). The transconjugants were checked for deletion of the whole fim3locus by PCR using oligonucleotides SPfim3UP2 and ASPfim3LO2.Reintroducing the whole fim3 locus with the 14 C stretch in the promoterwas done as follows. A 911-bp synthetic gene encompassing the wholelocus with the 14 C stretch was synthesized by GeneArt® Gene Synthesis(ThermoFisher SCIENTIFIC). SphI sites at the extremities of thesynthetic fragment were used to insert it into the SphI site ofpSKfim3UPLO giving rise to pSKfim3+. The correct orientation of theinsert was checked by restrictions. The 2256-bp SacI-BamHI fragment ofthis plasmid was transferred into the same restriction sites ofpJQ200mp18rpsL leading to pJQfim3+. This plasmid was used to do thedouble homologous recombination to obtain BPZE1f3. The recombinantstrain was verified by PCR using oligonucleotides SPfim3UP2 andASPfim3LO2.

Analysis of Fim2 and Fim3 Production

The B. pertussis strains were first inactivated by heating at 56° C. for30 minutes. The heat-inactivated strains were then coated at an opticaldensity (OD) 600 nm of 0.075 in 96-well plates (Nunc Maxi Sorp,) andincubated overnight at 37° C. until the wells were dry. The wells werethen blocked with 100 μl of PBS Tween 0.1% (PBST), containing 1% ofBovine Serum Albumin (BSA). Fim2 and Fim3 monoclonal antibodies (NIBSC,04/154 and 04/156, respectively) were added in serial dilutions from1/50 to 1/36450 in PBST (v/v). After three washes, the plates wereincubated with 100 μl of horseradish-peroxidase-labeled goat anti-mouseIgG (Southern Biotech) in PBST. Following five washes, the plates wereincubated with 100 μl of HRP Substrate TMB solution (Interchim) for 30min at room temperature. The reaction was stopped by the addition of 50μl of 1 M H₃PO₄. The OD was measured with a Biokinetic reader EL/340microplate at 450 nm.

DNA Sequencing

PCR amplification of chromosomal DNA was performed using PhusionHigh-Fidelity DNA Polymerase (Thermofisher) or KAPA HiFi DNA Polymerase(Kapa Biosystems) according to the manufacturer's instructions. The PCRfragments were purified with a QiaQuick PCR purification kit (Qiagen)and sequenced with the primers used for amplification. Primers ptxP Upand ptxP Low used for PCR amplification of ptxP have been describedpreviously (Mooi et al., Emerg Infect Dis 2009; 15:1206-13). Primers pmAF and pm AR used for partial PCR amplification of pm has been describedpreviously (Mooi et al., Infect Immun 1998; 66:670-5). Primers fim2 Up5′-AGCTAGGGGTAGACCACGGA-3′ and fim2 Low 5′-ATAACTCTTCTGGCGCCAAG-3′ wereused for amplification and sequencing of fim2. Primers fim3 Up5′-CATGACGGCACCCCTCAGTA-3′ and fim3 Low 5′-TTCACGTACGAGGCGAGATA-3′ wereused for amplification and sequencing of fim3.

Mouse Infection Experiments

BALB/c mice were obtained from Charles River (l'Abresle, France) andmaintained under specific pathogen-free conditions in the animalfacilities of the Institut Pasteur de Lille. Six week-old BALB/c micewere lightly sedated by intraperitoneal injection with an anestheticcocktail (ketamine+atropine+valium) before intranasal (i.n.)administration with 20 μl PBS containing 10⁶ colony-forming units (CFU)of B. pertussis BPZE1 or BPZElf3, as previously described (Mielcarek etal., PLoS Pathog 2006; 2:e65). Three mice per group were sacrificed atselected time points after i.n. administration, and their lungs wereharvested, homogenized in PBS and plated in serial dilutions ontoBG-blood agar to count CFUs after incubation at 37° C. for three to fourdays.

Mouse Protection Experiments

Six week-old BALB/c mice were i.n. vaccinated with 10⁵ CFU of B.pertussis BPZE1 or BPZElf3, as described above. Four weeks later, naïveand vaccinated mice were challenged with 10⁶ CFU of B. pertussis BPSM,the indicated clinical B. pertussis isolates or B. parapertussis in 20μl of PBS. Lung colonization was determined 3 h and 7 days later with 3and 5 mice per group, respectively.

Stability of Fim3 and Fim2 Production

10⁶ CFUs of BPZElf3 were administered to a sedated mouse in 20 μl ofPBS. 14 days later, the lung was harvested, homogenized and plated ontoBG agar. 3-4 days later, 94 individual colonies were inoculated into a96-well plate containing 100 μl of PBS/well. Control wells containedBPZE1, as a negative control, and BPZElf3 as a positive control. Theamount of bacteria present in each well was determined by OD measurementat 630 nm. After drying, the presence of Fim3 and of Fim2 was evaluatedby whole-cell ELISA as described above. After a blocking step with 100μl PBST containing 1% BSA, bacteria were incubated during one hour withthe anti-Fim3 monoclonal antibody 04/156 or anti-Fim2 monoclonalantibody 04/154 at a 1/1350 dilution in 100 μl PBST. After washes andincubation with 100 μl of horseradish-peroxidase-labeled goat anti-mouseIgG (Southern Biotech) in PBST, the presence of Fim3 or Fim2 wasevaluated with 100 μl of HRP Substrate TMB solution (Interchim)revelation. The reaction was stopped by the addition of 50 μl of 1 MH₃PO₄. The OD was measured with a Biokinetic reader EL/340 microplate at450 nm.

Results

Construction of BPZElf3.

In order to construct a BPZE1 derivative that produces Fim3, the fim3gene was first deleted from BPZE1. The upstream and downstream flankingregions of fim3 were amplified by PCR using the BPZE1 chromosomal DNA astemplate and were spliced together in the non-replicative vectorpJQ200mp 18rpsL (Antoine, J. Mol. Biol. (2005) 351, 799-809). The fim3gene of BPZE1 was then deleted by allelic exchange after conjugationwith E. coli SM10 containing the recombinant plasmid. The resultingstrain BPZE1Afim3 was used to re-integrate the fim3 gene together with afunctional promoter into the original fim3 locus. The 13-C stretch ofthe original promoter was replaced by a 14-C stretch, allowing for fim3expression and inserted into pSKfim3UPLO together with the fim3 openreading frame. The resulting plasmid pJQFim3+ was conjugated intoBPZE1Afim3 via conjugation with E. coli SM10: pJQFim3+. This resulted inBPZE1f3.

The production of Fim2 and Fim3 in BPZE1f3 was analyzed by whole-cellELISA using Fim2-specific and Fim3-specific monoclonal antibodies,respectively. As shown in FIG. 1A, both BPZE1 and BPZE1f3 producedequivalent amounts of Fim2. In contrast, Fim3 was only produced byBPZE1f3, and only background absorbency was detected with theFim3-specific antibody on whole BPZE1 extracts (FIG. 1B). Both strainsgrew equally well in Stainer Scholte medium and in the completelysynthetic Thijs medium (FIG. 2), indicating that the production of Fim3did not affect the growth characteristics of BPZE1f3.

Mouse Colonization by BPZE1f3.

To assess the potential role of Fim3 production by BPZE1f3 in thecolonization of the mouse respiratory tract, adult mice were infectedwith 10⁶ CFU of either BPZE1 or BPZE1f3, and 3 mice per group weresacrificed at days 3, 7, 14, 21 and 28 post-infection to quantify thebacterial loads in their lungs. As shown in FIG. 3, the capacity tocolonize the mouse lungs was identical between the two strains at alltime points analyzed, indicating that the production of Fim3 did notenhance, nor hamper the ability of BPZE1 to colonize the murinerespiratory tract.

BPZE1- and BPZE1f3-Mediated Protection Against Clinical B. pertussisIsolates.

To examine the relative protective effects of BPZE1 and BPZE1f3 againstclinical isolates that differ with respect to their production of Fim2and Fim3, we used a sub-optimal immunization protocol, in which micewere intranasally immunized with 10⁵ CFU of the vaccine strains andinfected one month later with 10⁶ CFU of the challenge strains. Thisprotocol was used because it is best suited to detect potentialdifferences between vaccine lots, as the standard vaccination protocolusing 10⁶ CFU of the vaccine strain followed two months later byinfection with 10⁶ CFU of the challenge strain usually results in totalclearance 7 days after challenge.

The potency of the two vaccine strains was tested against four differentclinical isolates from the B. pertussis culture collection of the RIVM(Bilthoven, The Netherlands). The five strains had the followingcharacteristics with respect to Fim2 and Fim3 production: 1617F1(Fim2+Fim3−), 403pF1 (Fim2+Fim3−), P134 (Fim2−Fim3−), 1412pF1(Fim2−Fim3+) and 403pF3 (Fim2+Fim3+). The genomic key features of thesestrains are presented in table I below. After vaccination and challengeinfection the bacterial load of the challenge strain was measured in thelungs 3 h and 7 days after infection.

TABLE I Key genomic features of the B. pertussis strains. Pptx¹ fim2fim3 serotype Prn³ ptx-s1⁴ BPSM P1 fim2-1² fim3-1² 2+/3− prn-1 ptxA2BPZE1 P1 fim2-1 fim3-1 2+/3− prn-1 ptxA2 (R9K, E129G) BPZE1f3 P1 fim2-1fim3-1 2+/3+ prn-1 ptxA2 (R9K, E129G) B1412 pF1 P1 fim2-1 fim3-1 2−/3+prn-1 ptxA1 B1617 pF1 P1 fim2-1 fim3-1 2+/3− prn-1 ptxA1 B0403 pF1 P1fim2-1 fim3-1 2+/3− prn-1 ptxA2 B0403 pF3 P1 fim2-1 fim3-1 2+/3+ prn-1ptxA2 P134S P1 fim2-1 fim3-1 2−/3− prn-1 ptxA2 ¹Promoter type of thepertussis toxin gene. ²Fimbrial gene genotype ³Pertactin gene allele⁴Pertussis toxin subunit S1 allele

BPZE1 and BPZE1f3 protected equally well against 1617 pF1, 403pF1, P134and 403pF3, diminishing the bacterial loads in each case by 4 to 5 logsat 7 days post-infection, compared to the bacterial loads innon-vaccinated mice (FIG. 4). There was no statistically significantdifference between BPZE1-vaccinated and BPZE1f3-vaccinated mice.However, when the mice were challenged with 1412pF1, the strain thatonly produces Fim3 and not Fim2, BPZE1f3 appeared to protectsignificantly better than BPZE1 (FIG. 4D). Whereas BPZE1 vaccinationresulted in a 4 log difference in bacterial load compared tonon-vaccinated mice, BPZE1f3 increased this protection to a 5 logdifference. No statistically significant decrease in bacterial loadsbetween vaccinated and non-vaccinated mice was observed when the CFUwere measured 3 h after challenge infection, indicating that, asexpected, all the mice had received the same challenge dose. Theseresults indicate improved potency of BPZElf3 over BPZE1 against strainsthat only produce Fim3, whereas there is no improvement in protectionagainst strains that produce Fim2 with or without Fim3, or againststrains that do not produce fimbriae.

BPZE1- and BPZElf3-Mediated Protection Against Bordetella parapertussis.

The potency of BPZElf3 against B. parapertussis was also tested. In thiscase, 10⁶ CFU of the vaccine strain was used, followed by challenge with10⁶ CFU of B. parapertussis two months after vaccination. It waspreviously shown that this protocol leads to strong protection, althoughnot to total clearance 7 days after challenge infection (Mielcarek etal., PLoS Pathog 2006; 2:e65). Seven days after B. parapertussisinfection, both BPZE1- and BPZElf3-vaccinated mice showed a strongreduction in bacterial load in the lungs (between 4 and 5 logs.)compared to non-vaccinated mice (FIG. 5). No statistical difference wasseen between BPZE1- and BPZE1f3-vaccinated mice, indicating that theproduction of Fim3 does not offer an advantage, nor is it detrimentalfor protection against B. parapertussis infection.

Stability of Fim3 Production by BPZElf3.

Since the only genetic difference between BPZE1 and BPZElf3 is theamount of C in the C-string of the fim3 promoter (13 C in BPZE1 and 14 Cin BPZElf3), and since C strings are prone to phase shift variation inB. pertussis (Willems et al., EMBO J 1990; 9:2803-9), the stability ofboth Fim3 and Fim2 production by BPZElf3 was evaluated after in vivopassaging of the vaccine strain in mice. Mice were infected with 10⁶ CFUof BPZElf3, and the bacteria present in the lungs 14 days afterinfection were harvested and plated onto BG agar. After growth, 94individual colonies were inoculated into a 96-well plate. The remainingcolonies were harvested and administered to mice for a second passage,followed 2 weeks later by a third passage. At each passage 94 individualcolonies were inoculated into a 96-well plate containing 1000 ofPBS/well. Control wells contained BPZE1, as a negative control, andBPZElf3 as a positive control. The amount of bacteria present in eachwell was determined by OD measurement at 630 nm. After drying, thepresence of Fim3 and Fim2 was evaluated by whole-cell ELISA. 94 of the94 clones were found to produce both Fim3 and Fim2 after the firstpassage. After the second passage 97.9% of the colonies produced Fim2and 96.8% produced Fim3, and after the third passage the numbers were87.23% and 97.9% for Fim3 and Fim2, respectively (FIG. 6), indicating arelatively stable fim3 expression, with only 12.77% loss after 3 in vivopassages.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A vaccine comprising a pharmaceuticallyacceptable carrier and a live attenuated Bordetella strain engineered tostably produce Fim3, wherein the live attenuated Bordetella strainretains the ability to colonize a mammalian subject's lungs and induce aprotective immune response against Bordetella infection.
 2. The vaccineof claim 1, wherein the live attenuated Bordetella strain stablyproduces Fim2.
 3. The vaccine of claim 1, wherein the live attenuatedBordetella strain has been rendered deficient in at least one virulencefactor selected from the group consisting of a functional pertussistoxin (PTX), a functional dermonecrotic toxin (DNT), and a functionaltracheal cytotoxin (TCT).
 4. The vaccine of claim 1, wherein the liveattenuated Bordetella strain has been rendered deficient in at least twovirulence factors selected from the group consisting of a functionalPTX, a functional DNT, and a functional TCT.
 5. The vaccine of claim 1,wherein the live attenuated Bordetella strain has been rendereddeficient in a PTX, a functional DNT, and a functional TCT.
 6. Thevaccine of claim 2, wherein the live attenuated Bordetella strain hasbeen rendered deficient in at least one virulence factor selected fromthe group consisting of a functional PTX, a functional DNT, and afunctional TCT.
 7. The vaccine of claim 2, wherein the live attenuatedBordetella strain has been rendered deficient in at least two virulencefactors selected from the group consisting of a functional PTX, afunctional DNT, and a functional TCT.
 8. The vaccine of claim 2, whereinthe live attenuated Bordetella strain has been rendered deficient in afunctional PTX, a functional DNT, and a functional TCT.
 9. The vaccineof claim 1, wherein the vaccine is provided in a single dosage formwhich comprises at least 1×10⁶ colony forming units (CFU) of the strain.10. The Bordetella strain designated BPZE1f3 deposited with theCollection Nationale de Cultures de Microorganismes (CNCM) underRegistration No. CNCM I-5247.
 11. A method of protecting a mammaliansubject from developing pertussis, the method comprising the step ofadministering to the mammalian subject a vaccine comprising apharmaceutically acceptable carrier and a live attenuated Bordetellastrain engineered to stably produce Fim3, wherein the live attenuatedBordetella strain retains the ability to colonize the mammaliansubject's lungs and induce a protective immune response againstBordetella infection.
 12. The method of claim 11, wherein the liveattenuated Bordetella strain stably produces Fim2.
 13. The method ofclaim 11, wherein the live attenuated Bordetella strain has beenrendered deficient in at least one virulence factor selected from thegroup consisting of a functional PTX, a functional DNT, and a functionalTCT.
 14. The method of claim 11, wherein the live attenuated Bordetellastrain has been rendered deficient in at least two virulence factorsselected from the group consisting of a functional PTX, a functionalDNT, and a functional TCT.
 15. The method of claim 11, wherein the liveattenuated Bordetella strain has been rendered deficient in a functionalPTX, a functional DNT, and a functional TCT.
 16. The method of claim 12,wherein the live attenuated Bordetella strain has been rendereddeficient in at least one virulence factor selected from the groupconsisting of a functional PTX, a functional DNT, and a functional TCT.17. The method of claim 12, wherein the live attenuated Bordetellastrain has been rendered deficient in at least two virulence factorsselected from the group consisting of a functional PTX, a functionalDNT, and a functional TCT.
 18. The method of claim 12, wherein the liveattenuated Bordetella strain has been rendered deficient in a functionalPTX, a functional DNT, and a functional TCT.
 19. The method of claim 11,wherein the strain is BPZE1f3 deposited with the Collection Nationale deCultures de Microorganismes (CNCM) under Registration No. CNCM I-5247.